Successful development of new approaches for the immunotherapy of cancer requires an understanding of complex, interdependent activities of early innate response elements with subsequent powerful adaptive immune responses. We are taking several novel approaches to maximize the host's ability to mount an effective antitumor response. These approaches include optimizing antigen presenting capability through the ligation of CD40, a TNF superfamily receptor that serves as a potent trigger for dendritic cells and macrophages, which provide a key interface between innate and adaptive responses. The potency of dendritic cell stimulation by agonist CD40 antibodies is enhanced when used in conjunction with IL-2 or IL-15 and the combination of agonist anti-CD40 plus either IL-2 or IL-15 shows enhanced antitumor activity against metastatic kidney cancer in mice. We have analyzed leukocytes contained within the primary tumors that develop in the kidney as well as those in the lungs and livers, which serve as primary and secondary sites of tumor metastases, respectively. After treating tumor-bearing mice with IL-2/anti-CD40 or IL-15/anti-CD40, we have identified the recruitment of macrophages and T cells that appear to be critical mediators of anti-tumor responses. Once present at the developing tumor site, these cells are capable of producing many different soluble mediators, such as interferon gamma (IFNg), nitric oxide, and VEGF that may influence tumor progression. By regulating the ability of these cell types to accumulate within tumors, we are identifying the role that these cells play during both primary tumor progression and metastasis to distant organs. Our data have illustrated the potential for dramatic mechanistic differences in biological effects mediated by anti-CD40 alone versus its use in combination with IL-2 that includes the synergistic upregulation of IFNg and nitric oxide expression that controls tumor burden. To date, we have shown IL-2/anti-CD40 induces enhanced antitumor responses that depend on the infiltration of established tumors by effector CD8+ T cells and a concomitant IFNg-dependent reduction in CD4+/FoxP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC) and Th2 chemokine expression within the tumor-microenvironment. These results may help to explain the limited clinical efficacy of anti-CD40 as a single agent based on its inability to remove Tregs and MDSC specifically from within the tumor microenvironment and they suggest that anti-CD40 may be more beneficial in combination with other selected immune agents, such as IL-2. Our ongoing work is investigating the mechanisms underlying the selective loss of regulatory T cells and MDSC within the tumor microenvironment. Our data indicates that IL-2/anti-CD40 induces the expression of Fas on these cell populations, as well as the infiltration of primary tumors by Fas ligand expressing CD8+ T cells and other leukocytes. This process elicits Fas-dependent cell death, or apoptosis, of Tregs and MDSC. Our data demonstrates the critical importance of Fas-mediated Treg and MDSC removal towards the anti-tumor efficacy of IL-2/anti CD40 combination therapy. Furthermore, they suggest that immunotherapeutic strategies, such as IL-2/anti CD40, that target the susceptibility of Tregs and MDSC to Fas-mediated cell death hold promise for further development as cancer treatment strategies. Our current and future studies seek to clarify the cellular and molecular events critical for the observed biological effects of aCD40, and the potential for complementary use of anti-CD40 with rationally selected molecularly targeted agents. In one such approach, we have combined anti-CD40 with an ATP competitive mTOR drug, AZD8055, developed by AstraZeneca. The rationale for this strategy is two-fold. First, mTOR inhibition represents is currently a leading clinical target for RCC. We also hypothesized that combining AZD8055 with aCD40 antibody would induce more efficient antitumor effects by a combination of direct tumor killing and subsequent release of tumor-associated antigens to antigen presenting cells and coincident modulation of immune cell functions in vivo. The results of our recently published study show that in a syngeneic mouse metastatic renal cell carcinoma (RCC) model, AZD8055 and aCD40 synergize for tumor regression by activating macrophages and DCs and inducing strong Th1 immune responses in the tumor microenvironment. We have also translated our earlier preclinical findings using IL-2 in combination with IL-12 to human clinical trials. We have completed a phase I clinical trial of IL-12/pulse IL-2 at the NIH Clinical Research Center via collaboration with Drs. Jon Wigginton and John Janik (now at BMS). IL-2 was administered every 8 hours on days 1 and 9 and intravenous IL-12 was administered once daily on days 2, 4, 6, 10, 12 and 14 of each 35 day cycle of therapy. The maximum tolerated dose was determined to be 600,000 IU/kg IL-2 and 300 ng/kg IL-12. Anticipated clinical side effects of fever, chills, headache, fatigue, hypoxia, hypotension, myalgias, nausea, vomiting and stomatitis, neutropenia and lymphopenia were seen in most patients. Toxicities generally resolved promptly upon completion of each cycle of therapy. IFN-gamma production peaked after the first IL-12 administration in comparison with subsequent doses. Interestingly, IL-2 partially rescued IFN-gamma production following IL-12 injection in the second week of therapy, and IL-18 levels and IL-12 receptor (CD212Beta1) both increased following IL-12/pulse IL-2 therapy. Regression of individual tumor lesions was seen in many patients, and two patients with RCC achieved objective partial responses. Although IL-2 partially overcame IL-12 induced tachyphylaxis and induced regression of some lesions, the overall objective response rate was not improved over IL-12 or IL-2 as single agents. One possible explanation for this effect could be the inability of exogenous systemic cytokine administration to achieve levels needed in the tumor microenvironment to re-structure local immune components, and this hypothesis formed part of the basis for our current use of IL-2 in combination with anti-CD40 which, among its diverse effects in the tumor microenvironment, induces IL-12 production by CD40 positive leukocytes.